SUMOs (small ubiquitin-like modifiers) are ubiquitin-like proteins (Ubls) that become conjugated to substrates through a pathway that is biochemically similar to ubiquitination. SUMOylation is involved in many cellular processes, including DNA metabolism, gene expression and cell cycle progression. Clinical studies suggest that SUMOylation plays important roles in disease processes, including diabetes, viral infection and carcinogenesis. Notably, there are a wide variety of SUMOylated target proteins, many of whose modification may be mis-regulated in human diseases. While SUMOylation is thus broadly implicated in the pathology of these diseases, one major challenge in this field is to understand its role at the level of individual substrates and of the cellular processes that they regulate. We are particularly interested in the capacity of SUMOylation to control cell division and nuclear transport. Vertebrate cells express three major SUMO paralogues (SUMO-1-3): Mature SUMO-2 and -3 are 95% identical to each other, while SUMO-1 is 45% identical to SUMO-2 or -3. (Where they are functionally indistinguishable, SUMO-2 and -3 will be collectively called SUMO-2/3.) Like ubiquitin, SUMO-2/3 can be assembled into polymeric chains through the sequential conjugation of SUMOs to each other. SUMO-1 appears less likely to form similar chains, and might only become conjugated to the end of SUMO-2/3 chains as a chain terminator. A large number of SUMOylation substrates have been identified. SUMOylation promotes a variety of fates for individual targets, dependent upon protein itself, the conjugated paralogue and whether the conjugated species contains a single SUMO or SUMO chains. SUMOylation is a dynamic process because SUMO proteases rapidly turn over conjugated species. The major enzymes that catalyze both SUMO processing and deSUMOylation are members of the same family of proteases, called Ubl specific proteases (Ulp) in yeast and Sentrin-specific proteases (SENP) in vertebrates. There are two budding yeast Ulps (Ulp1p and Ulp2p/Smt4p), and six mammalian SENPs (SENP1, 2, 3, 5, 6, and 7). Ulp1p localizes to nuclear pore complexes (NPCs) and catalyzes both processing and deconjugation of the yeast SUMO protein. Ulp1p is required for G2/M progression during the cell cycle, and it is encoded by an essential gene. We previously found that frogs possess a single Ulp1p-like protease, xSENP1, which binds nuclear pores and may perform equivalent functions. Our data indicate that xSENP1 is important for mitotic exit in embryonic systems, such as Xenopus egg extracts, and that it may regulate proteolysis through proteasomes. Human cells have two Ulp1p-like proteases, SENP1 and SENP2, which localize to NPCs, and catalyze processing and deconjugation of all SUMO paralogues. We are investigating these proteins in order to understand SUMO pathway regulation in mammalian somatic systems. The first question we are seeking to address concerns the interactions that target SENP1 and SENP2 to NPCs. Earlier reports have indicated that SENP2 targeting occurs through association with the Nup107-160 NPC subcomplex, using a mechanism that is dependent on the nuclear transport receptor karyopherin-alpha. At the same time, a unique N-terminal domain of the nucleoporin Nup153 and domain within its C-terminal FG-rich region have likewise been implicated in SENP1 and SENP2 localization at the NPC. To clarify the nature of these SENP-nucleoporin interactions, we are using CRISPR/Cas9-based gene editing to tag individual nucleoporins with an auxin-induced degron (AID), allowing their regulated and rapid destruction. We are currently identifying nucleoporins whose degradation results in SENP1 and SENP2 release from NPCs, and the consequence of this release upon global SUMOylation. The second question we are seeking to address concerns whether SENP1 and SENP2 control distinct events or act in a manner that is fully redundant, using CRISPR/Cas9-based gene editing to make knockout or AID-tagged lines for each protein. We are analyzing the phenotypic consequences of depletion of these enzymes, individually or simultaneously, as well as their consequences for global SUMOylation, cell cycle progression and gene expression patterns.